WO2014202616A2 - Gène de rasamsonia et son utilisation - Google Patents
Gène de rasamsonia et son utilisation Download PDFInfo
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- WO2014202616A2 WO2014202616A2 PCT/EP2014/062737 EP2014062737W WO2014202616A2 WO 2014202616 A2 WO2014202616 A2 WO 2014202616A2 EP 2014062737 W EP2014062737 W EP 2014062737W WO 2014202616 A2 WO2014202616 A2 WO 2014202616A2
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
Definitions
- the present invention relates to DNA of Rasamsonia emersonii.
- This DNA contains nucleotide sequences such as genes, regulatory elements and other nucleotide sequences.
- the present invention also relates to polypeptides having biological activity and comprising an amino acid sequence encoded by a nucleotide sequence.
- the present invention relates to DNA of Rasamsonia emersonii.
- This DNA contains genes, regulatory elements and other nucleotide sequences.
- This DNA may encode useful biologically active substances such as enzymes. These enzymes may be used in several industrial applications. Enzymes are for example used in the chemical industry and other industrial applications when specific catalysts are required, for example in the food or feed, textile, pulp or paper or detergent industries and other industries. Enzymes in general are limited in the number of reactions they have evolved to catalyze and their deactivation at high temperatures. As a consequence, protein engineering is an active area of research and involves attempts to create new enzymes with novel properties, either through rational design or in vitro evolution. These efforts have begun to be successful, and a few enzymes have now been designed to improve enzymatic reactions. For designing it is essential to have starting sequences from useful microorganisms especially thermophilic microorganisms like fungi.
- the present invention provides a polypeptide which comprises
- variant polypeptide has at least 70% sequence identity with the sequence set out in SEQ ID NO: 20501 to 32240, or (ii) has an amino acid sequence that differs in 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 or 12 amino acids from the amino acid sequence of SEQ ID NO: 20501 to 32240.
- the polypeptide of the invention has biological activity, preferably is a polypeptide such as an enzyme, more preferably is an oxidoreductase, transferase, hydrolase, lyase, isomerase or ligase or is capable to alter or influence the expression of an oxidoreductase, transferase, hydrolase, lyase, isomerase or ligase and most preferably is a carbohydrate degrading enzyme and/or carbohydrate hydrolysing enzyme, preferably has an activity mentioned in Table 1 .
- the present invention provides a polynucleotide having a nucleic acid sequence whereby the nucleic acid sequence is selected from the group consisting of:
- nucleic acid sequence encoding (i) the amino acid sequence of SEQ ID NO: 20501 to 32240, (ii) an amino acid sequence having at least 70% identity with the amino acid sequence of SEQ I D NO: 20501 to 32240, or (iii) an amino acid sequence that differs in 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 or 12 amino acids from the amino acid sequence of SEQ ID NO: 20501 to 32240; or
- the polynucleotide of the invention has a nucleic acid sequence coding for a polypeptide which has biological activity
- the polypeptide is a polypeptide such as an enzyme, more preferably is an oxidoreductase, transferase, hydrolase, lyase, isomerase or ligase or is capable to alter or influence the expression of an oxidoreductase, transferase, hydrolase, lyase, isomerase or ligase and most preferably is a carbohydrate degrading enzyme and/or carbohydrate hydrolysing enzyme, preferably has an activity mentioned in Table 1 .
- the invention also provides a nucleic acid construct or vector comprising the polynucleotide of the invention and a cell comprising the polynucleotide of the invention or a nucleic acid construct or vector of the invention.
- the invention further provides a cell wherein the polynucleotide according to the invention is mutated or deleted from the genome to obtain lower or no activity of the polypeptide or lower or no expression of the polypeptide encoded by said polynucleotide compared to the cell wherein the polynucleotide of the invention is not mutated or deleted from the genome.
- the invention further provides a mutant or recombinant cell wherein the polynucleotide according to the invention is lower or not expressed compared to the native or starting cell. Lower expression can be obtained for example by altering or deleting the promoter.
- the invention further provides a mutant or recombinant cell wherein the polynucleotide according to the invention is overexpressed compared to the native or starting cell.
- Overexpression can be obtained for example by altering the promoter or by introducing one or more copies of polynucleotide according to the invention in the cell.
- the cell of the invention is a fungal cell, preferably a fungal cell selected from the group consisting of the genera Acremonium, Agaricus, Aspergillus, Aureobasidium, Chrysosporium, Coprinus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Piromyces, Panerochaete, Pleurotus, Schizophyllum, Talaromyces, Rasamsonia, Saccharomyces, Thermoascus, Thielavia, Tolypocladium, and Trichoderma.
- the cell of the invention is a recombinant cell.
- one or more gene can be deleted, knocked-out or disrupted in full or in part, wherein optionally the gene encodes for a protease.
- the invention also provides a method for the preparation of a polypeptide according to the invention having preferably biological activity, which method comprises cultivating a cell of the invention under conditions which allow for expression of said polypeptide and, optionally, recovering the expressed polypeptide.
- the invention also provides a method of producing an enzyme or enzyme composition comprising the fermentation of the cell according of the invention and optionally recovery of the enzyme or enzyme composition
- the invention provides a composition
- a composition comprising: (i) the polypeptide of the invention and; (ii) a cellulase and/or a hemicellulase and/or a pectinase, preferably the cellulase is a GH61 , cellobiohydrolase, cellobiohydrolase I, cellobiohydrolase II, endo- ⁇ -1 ,4-glucanase, ⁇ -glucosidase or ⁇ -(1 ,3)(1 ,4)-glucanase and/or the hemicellulase is an endoxylanase, ⁇ -xylosidase, oL-arabinofuranosidase, oD-glucuronidase feruloyl esterase, coumaroyl esterase, a-galactosidase, ⁇ -galactosidase, ⁇ -mannanase or ⁇ - mannosidas
- Another aspect of the invention relates to the use of a polypeptide of the invention and/or a composition of the invention to produce sugar from a lignocellulosic material.
- the invention also provides:
- a method for the preparation of a polypeptide having preferably biological activity comprises cultivating a cell of the invention under conditions which allow for expression of said polypeptide and, optionally, recovering the expressed polypeptide;
- composition comprising: (i) a polypeptide of the invention and; (ii) a cellulase and/or a hemicellulase and/or a pectinase.
- polypeptides of the invention may have all kinds of use.
- the polypeptides of the invention having carbohydrate degrading and/or carbohydrate hydrolysing activity may be used in industrial processes.
- the invention provides a method for the treatment of a substrate comprising carbohydrate material which method comprises contacting the substrate with a polypeptide or a composition of the invention.
- a method for producing a sugar or sugars from lignocellulosic material comprises contacting the lignocellulosic material with a polypeptide or a composition of the invention.
- the invention provides a method for producing a fermentation product, which method comprises: producing a fermentable sugar using the described above; and fermenting the resulting fermentable sugar, thereby to produce a fermentation product.
- a polypeptide or a composition of the invention may also be used, for example, in the preparation of a food product, in the preparation of a detergent, in the preparation of an animal feed, in the treatment of pulp or in the manufacture of a paper or in the preparation of a fabric or textile or in the cleaning thereof.
- the invention also provides: a processed material obtainable by contacting a plant material or lignocellulosic material with a polypeptide or a composition of the invention;
- a food or feed comprising a polypeptide or a composition of the invention; and a plant or a part thereof which comprises a polynucleotide, a polypeptide, a vector or a cell according to the invention.
- the invention further provides a polynucleotide coding for a signal sequence having a sequence set out in SEQ ID NO: 32241 to 33730 or having a sequence that differs 1 , 2 or 3 amino acids from the sequence set out in SEQ ID NO: 32241 to 33730.
- Fig 1 Map of pGBTOP for expression of genes in A. niger. Depicted are the gene of interest (GOI) expressed from the glucoamylase promoter (PglaA). In addition, the glucoamylase flank (3'glaA) of the expression cassette is depicted. In this application a gene of interest is the coding sequence of the protein or polypeptide of the invention as defined hereinafter.
- Fig. 2 shows a schematic diagram of plasmid pEBA1006 that was used in bipartite gene-targeting method in combination with the pEBA expression vector containing the protein of the invention with the goal to replace the RePepA ORF and approximately 1500 nucleotides upstream of the start ATG codon by the expression cassette of the protein of the invention in Rasamsonia emersonii.
- the vector comprises the 3' part of the ble coding region, the A.nidulans trpC terminator, a lox71 site, a 2500 bp 3' flanking region of the RePepA ORF, and the backbone of pUC19 (Invitrogen, Breda, The Netherlands).
- the £. coli DNA was removed by digestion with restriction enzyme A/oil, prior to transformation of the R. emersonii strains.
- Fig. 3 shows a schematic diagram of pEBA expression plasmid containing the protein of the invention that was used in bipartite gene-targeting method in combination with the pEBA1006 vector with the goal to replace the RePepA ORF and approximately 1500 nucleotides upstream of the start ATG codon by the expression cassette of the protein of the invention in Rasamsonia emersonii.
- the vector comprises a 1500 bp 5' flanking region 1 .5 kb upstream of the RePepA ORF for targeting in the RePepA locus, the protein of the invention-expression cassette consisting of R.
- emersonii promoter 2 the protein of the invention-coding region and the A.nidulans amdS terminator (TamdS), a lox66 site, the non-functional 5' part of the ble coding region (5' ble) driven by the A.nidulans gpdA promoter.
- the E. coli DNA was removed by digestion with restriction enzyme A/oil, prior to transformation of the R. emersonii strains.
- Fig. 4 shows the pEBADEL.1 vector.
- Part of the vector fragment is used in bipartite gene-targeting method in combination with the pEBADEL2 vector with the goal to delete the ORF of the protein of the invention (GOI) in Rasamsonia emersonii.
- the vector comprises a 1200 bp 5' upstream flanking region of the ORF encoding the protein of the invention and the 5' part of the ble coding region driven by the A.nidulans gpdA promoter and the backbone of pUC19 (Invitrogen, Breda, The Netherlands).
- the E. coli DNA is removed by digestion with restriction enzyme Notl, prior to transformation of the R. emersonii strains.
- Fig. 5 shows the pEBADEL.1 vector.
- Part of the vector fragment is used in bipartite gene-targeting method in combination with the pEBADEL.1 vector with the goal to delete the ORF encoding the protein of the invention (GOI) in Rasamsonia emersonii.
- the vector comprises the 3' part of the ble coding region, the A.nidulans trpC terminator, and a -1200 bp 3' flanking region downstream of the ORF encoding the protein of the invention, and the backbone of pUC19 (Invitrogen, Breda, The Netherlands).
- the E. coli DNA is removed by digestion with restriction enzyme Notl, prior to transformation of the R. emersonii strains.
- Fig. 6 shows the strategy used to delete the ORF gene encoding the protein of the invention in R. emersonii.
- the deletion vectors comprise the overlapping non-functional ble selection marker fragments (split marker) flanked by loxP sites and 5' and 3' homologous regions of the gene encoding the protein of the invention (GOI) for targeting (1 ).
- the constructs integrate through triple homologous recombination (X) at the locus of the gene of the invention and at the overlapping homologous non-functional ble selection marker fragment (2) and replaces the genomic gene copy (3).
- Re01 .g01 190 hypothetical protein 1 16 10366 20616
- Re01 .g01330 Putative uncharacterized protein 130 10380 20630
- Beta-galactosidase EC 3.2.1.23 259 10509 20759 30788 32278
- Re01.g02930 Putative uncharacterized protein 290 10540 20790
- Re01.g02980 Putative uncharacterized protein 295 10545 20795
- Beta-glucosidase EC 3.2.1.21
- Re01.g03400 40S ribosomal protein S5 336 10586 20836 Re01.g03410 SDA1 domain protein 337 10587 20837
- Re01.g03560 (AFlLorthologue AFUAJ G15240) 352 10602 20852
- Re01.g04010 (AFU . orthologue AFUAJ G14380) 396 10646 20896
- Re01.g04170 protein 1 putative 412 10662 20912
- Re01.g04400 Remark: YS24 is part of the small 435 10685 20935
- Pre-rRNA processing protein Tsr1 Pre-rRNA processing protein
- Re01.g05320 protein putative 525 10775 21025
- Protein transport protein (SEC31 ),
- Lactoylglutathione lyase (Glo1 )
- Re01.g06580 Putative uncharacterized protein 650 10900 21 150 30842 32332
- Re01.g06590 Putative uncharacterized protein 651 10901 21 151 30843 32333
- Re01.g07080 Putative uncharacterized protein 700 10950 21200
- Hexokinase family protein XprF Hexokinase family protein XprF
- Rab6 of H. sapiens is
- Re01.g07680 Putative uncharacterized protein 759 1 1009 21259 30865 32355
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- Proteomics, Peptides & Aminoacids (AREA)
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Abstract
L'invention concerne un polypeptide qui comprend (a) une séquence d'acides aminés présentée dans SEQ ID NO: 20501 à 32240; (b) une séquence d'acides aminés codée par la séquence d'acide nucléique de SEQ ID NO: 1 à 20500; (c) ou un variant polypeptidique, le de polypeptidique (i) ayant au moins 70 % d'identité de séquence avec la séquence présentée dans SEQ ID NO: 20501 à 32240, ou (ii) ayant une séquence d'acides aminés qui diffère dans 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 ou 12 acides aminés à partir de la séquence d'acides aminés de SEQ ID NO: 20501 à 32240. L'invention concerne également des procédés d'utilisation du polypeptide dans des procédés industriels. L'invention concerne également des cellules transformées par un polynucléotide selon l'invention, appropriées pour la production de ces protéines.
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WO2016138315A1 (fr) * | 2015-02-25 | 2016-09-01 | Danisco Us Inc | Alpha-glucosidase, compositions et procédés |
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US20170101474A1 (en) * | 2015-10-13 | 2017-04-13 | Formurex, Inc. | Synthetic antibody mimic peptides |
WO2017140807A1 (fr) | 2016-02-16 | 2017-08-24 | Monaghan Mushrooms Group | Polypeptides fongiques à activité de lysozyme |
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